This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Phosporyl transfer reactions play a fundamental role in a wide range of biological processes such as basic metabolism, gene expression, and cell signaling, and enzymes that catalyze reactions at phosphorus have some of the largest rate accelerations known. A significant challenge in understanding the chemistry of biological phosphoryl transfer is in defining mechanisms through which phosphoryl transfer enzymes achieve their chemical selectivity. For example, alkaline phosphatase from E. coli (AP) and nucleotide pyrophosphatase/phosphodiesterase from X. axonopodis (NPP) both catalyze hydrolysis reactions of phosphate diesters and monoesters at a structurally very similar binuclear zinc active site ligated by His and Asp residues. However, AP preferentially catalyzes monoester hydrolysis by a factor of 10^9, whereas NPP preferentially catalyzes diester hydrolysis by factors ranging from 10^2 to 10^6. It has been suggested that fine structural differences between the binuclear Zn sites of AP and NPP that are beyond the resolution of the current x-ray crystallographic structures (1.7 ? 2.0 A) may account for some of the specificity for phosphate monoesters or diesters. We will use XAS to study the binuclear active center in AP and NPP for a variety of protein forms such as holoenzyme, substrate- (phosphate monoester or diester), inhibitor- (inorganic phosphate or phosphate monoester), and transition state analog (vanadate) bound enzymes. These studies will include wild-type zinc-containing AP and NPP as well as their cobalt-substituted analogs. By applying XAS technique at the Zn, Co, and V K-edge, we will be able to provide electronic and high-resolution structural information on the metal center during the catalytic cycle. When coupled to biochemical and kinetic data, these experiments will contribute to comparative structural analysis of AP and NPP and advance our understanding of how structural features contribute to chemical selectivity of the binuclear metal site.